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Series GSE5339 Query DataSets for GSE5339
Status Public on Jan 01, 2007
Title Vanadium pentoxide induced gene expression in human lung fibroblasts
Organism Homo sapiens
Experiment type Expression profiling by array
Summary Exposure to vanadium pentoxide (V2O5) is a cause of occupational bronchitis. We evaluated gene expression profiles in cultured human lung fibroblasts exposed to V2O5 in vitro in order to identify candidate genes that could play a role in airway remodeling associated with V2O5-induced bronchitis. Gene expression was measured at various time points over a 24 hr period using the Affymetrix Human Genome U133A 2.0 Array. Expression data were preprocessed using RMA with a log2 transformation. Statistical analysis was performed in R using the affylmGUI package using a linear model with contrasts between untreated control and V2O5-exposed fibroblasts. Genes identified as statistically significant were filtered by selecting only those genes that exhibited a > 2-fold change. Quantitative real-time RT-PCR was utilized to confirm expression of selected genes. More than 2000 genes were significantly changed in response to V2O5 over the time course of our experiment. Genes altered by V2O5 were involved in biologic processes related to cell growth and differentiation, oxidative stress responses, immune regulation, and interferon signaling and apoptosis. In particular, V2O5 induced genes that encode growth factors involved in epithelial repair (HB-EGF) or angiogenesis (VEGF), peroxide generating enzymes (SOD2), pro-inflammatory enzymes (PGHS2), while suppressing genes involved in growth arrest (GAS1, STAT-1) and cell cycle inhibition (CDKN1B). Our study also identified a variety of novel genes that could be used as biomarkers of V2O5-induced bronchitis or could serve as candidate genes for disease progression.
Keywords: time course, stress response
 
Overall design Experimental Design
Normal adult human lung fibroblasts (ATCC 16 Lu) were purchased from American Type Culture Collection (Rockville, MD). Fibroblasts were seeded into 175 cm2 plastic culture flasks and grown to confluence in 10% fetal bovine serum (FBS)/Dulbecco’s modified Eagle’s medium (DMEM), then trypsin-liberated, and seeded into 150 mm dishes. Confluent monolayers were rendered quiescent for 24 hrs in serum-free defined medium (SFDM) that consisted of Ham’s F-12 medium with 0.25% BSA with an insulin/transferrin/selenium supplement. Cells were treated with 10 µg/cm2 vanadium pentoxide, V2O5 (Aldrich Chemical, Milwaukee, WI) or SFDM and RNA was harvested from the fibroblast cultures at 1, 4, 8, 12 and 24 hrs post-treatment. RNA from an SFDM control was harvested at each of these time points to normalize the V2O5 treatment at the same corresponding time point. Three replicate chips were analyzed for SFDM and V2O5 treatment groups at each of the five time points tested.

Microarray Hybridizations and Data Analysis
Human lung fibroblast RNA was isolated using RNeasy columns (Qiagen, Valencia, CA). RNA quality was verified by spectrophotometry and gel electrophoresis using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Probe preparation and hybridization to the microarray was performed in the CIIT Gene Expression Core Facility using standard Affymetrix procedures. Double-stranded cDNA was synthesized from RNA using an oligo-dT24-T7. Biotinylated cRNA was synthesized from an aliquot of the cDNA template using the T7 RNA Transcript Labeling Kit (ENZO Diagnostics, Farmingdale NY). The labeled cRNA was then fragmented, hybridized to Affymetrix Human Genome U133A 2.0 arrays (Affymetrix, Santa Clara, CA), and stained using phycoerythrein-conjugated streptavidin (Molecular Probes, Eugene, OR).

Statistical Analysis and Data Processing
The microarray data were preprocessed using RMA with a log base 2 (log2) transformation. Statistical analysis of the data was performed in R using the affyImGUI package. To identify genes with significant changes in expression following V2O5 exposure, all treatment groups were analyzed using a linear model with contrasts between untreated fibroblasts and V2O5-exposed fibroblasts at each time point. Probability values were adjusted for multiple comparisons using a false discovery rate of 5% (FDR = 0.05). Genes identified as statistically significant were subject to an additional filter by selecting only those genes that exhibited a > 2-fold change from the untreated fibroblasts.
 
Contributor(s) Ingram JL, Menezes AA, Turpin EA, Mangum JB, Pluta LJ, Thomas RS, Bonner JC
Citation(s) 17459161
Submission date Jul 18, 2006
Last update date Dec 06, 2018
Contact name James C Bonner
E-mail(s) jbonner@ciit.org
Phone 919-558-1443
Fax 919-558-1300
Organization name CIIT Centers for Health Research
Department Biological Sciences
Street address 6 Davis Drive
City Research Triangle Park
State/province NC
ZIP/Postal code 27709
Country USA
 
Platforms (1)
GPL571 [HG-U133A_2] Affymetrix Human Genome U133A 2.0 Array
Samples (45)
GSM120300 cells_ATCC 16 Lu _human lung fibroblasts_JCB_0_12_19_Lung_Fibroblast_Rcontrol_HG-U133A_2_IMFRT0014
GSM120301 cells_ATCC 16Lu_human lung fibroblasts_JCB_0_12_34_Lung_Fibroblast_control_HG-U133A_2_IMFRT0014
GSM120302 cells_ATCC 16Lu_human lung fibroblasts_JCB_0_12_4_Lung_Fibroblast_control_HG-U133A_2_IMFRT0014
Relations
BioProject PRJNA96361

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